http://archive.rubicon-foundation.org
4
Diving and Hyperbaric Medicine Volume 40 No. 1 March 2010
Short communications
Variations in exhaled nitric oxide concentration after three types of
dive
Pieter-Jan van Ooij, Antoinette Houtkooper and Rob van Hulst
Key words
Diving, hyperbaric, hyperoxia, expired nitric oxide, FENO
Abstract
(van Ooij P-J, Houtkooper A, van Hulst R. Variations in exhaled nitric oxide concentration after three types of dive. Diving
and Hyperbaric Medicine. 2010;40(1):4-7.)
Introduction: An increase in exhaled nitric oxide concentration (FENO) occurs during an exacerbation of chronic obstructive
lung disease or other inflammatory processes of the airway. Raised FENO levels are also observed during normobaric, mild
hyperoxic exposures, whereas after hyperbaric hyperoxic exposure the FENO level is reduced. This study investigated the
variations of FENO after three different types of dive.
Methods: Military divers participated in either a closed circuit rebreather dive (CCR, n = 17, pO2 = 130 kPa), semi-closed
circuit rebreather dive (S-CCR, n = 12, pO2 = 180 kPa) or a compressed air dive (scuba, n = 17, pO2 = 126 or attendant,
n = 12, pO2 = 118 kPa). Before and after each dive, the FENO was measured using a hand-held electrochemical analyser
(Niox Mino®).
Results: All values for FENO fell within the normal range (5−25 ppb). A small decrease in FENO level was found after all
dives. After CCR dives, FENO fell from 16.4 (± 8.0) pre-dive to 13.6 (± 7.5) ppb, after S-CCR from 16.2 (± 7.2) to 13.6
(± 6.3) ppb, scuba from 17.1 (± 5.6) to 16.1 (± 5.2) ppb and attendants from 17.7 (± 9.8) to 17.3 (± 9.1) ppb. Only after a
CCR or S-CCR dive was this decrease statistically significant (P < 0.05).
Conclusion: In our divers, hyperbaric hyperoxia up to 180 kPa led to a small decrease in FENO in the conductive compartment
of the lungs, the biological importance of which is unknown.
Introduction
Nitric oxide (NO) is a small molecule with the qualities
of a free radical. In the human body, NO is made under
the influence of NO-synthase (NOS) from the oxidation
of L-arginine by NOS to L-citrulline with the release of
NO.1 Three forms of NOS are described: neuronal NOS
(nNOS), endothelial NOS (eNOS) and inducible NOS
(iNOS); nNOS and eNOS are often termed constitutional
NOS (cNOS).1,2 While cNOS is constantly available and
produces low quantities of NO, iNOS will produce NO in
large quantities under more extreme circumstances, such
as an inflammatory process.2 In the lung, cNOS is found
in the epithelium, endothelium and neurons, while iNOS is
found in the epithelium and macrophages. Despite its radical
qualities, the half-life of NO in air can be tens of seconds,
which allows us to measure NO in the exhaled breath.1
NO, in reaction with oxygen, can be metabolized to
nitrite (NO2-), nitrate (NO3-), and peroxynitrite (ONOO-).
Peroxynitrite has cell- and tissue-damaging activity which
causes inflammation. This kind of inflammation plays an
important role in the exacerbation of asthma or chronic
obstructive pulmonary disease (COPD).1 In 1899, LorrainSmith demonstrated that breathing oxygen at a partial pressure
(pO2) higher than 50 kPa can cause pulmonary damage,
leading to pulmonary oedema and airway inflammation.3 In
view of this inflammation, one would expect an increase in
the exhaled nitric oxide concentration (FENO) after exposure
to a breathing gas with a pO2 of more than 50 kPa. However,
earlier studies showed inconsistent results. Raised FENO
levels were observed during normobaric, mild hyperoxic
exposures,4,5 whereas after hyperbaric hyperoxic exposure
the FENO level was reduced.2,6 The aim of this study was
to investigate the effect on FENO of three different types
of dive, each using a different breathing gas with a pO2 of
more than 100 kPa.
Methods
STUDY POPULATION
All participating divers and attendants (n = 58) were
professional military divers and fit to dive. All dives used to
measure FENO were made as part of their daily routine or
as part of their training. Each diver or attendant participated
in only one type of dive. All participants were male and
were informed about the aims of the study during a general
meeting. It was explained that they could withdraw from the
study at any time and the FENO results would not be put in
their personal medical file. Before FENO measurement, they
were asked again if they had any questions or objections
regarding the study. Table 1 presents demographic data on
the divers and attendants.
http://archive.rubicon-foundation.org
Diving and Hyperbaric Medicine Volume 40 No.1 March 2010
5
Table 1
Demographic data, mean (SD) for the participating divers and attendants (n = 58)
Height (cm)
Weight (kg)
Age (years)
Smoking (pack-years)
CCR divers (n = 17)
181.4 (6.2)
85.6 (8.7)
24.5 (2.6)*
0
S-CCR divers
(n = 12)
184.8 (5.4)
91.7 (10.8)
34.3 (8.5)*
0
Scuba divers
(n = 17)
183.8 (5.9)
89.5 (11.6)
40.2 (6.5)†
0.9 (2.6)
Attendants
(n = 12)
181.5 (7.9)
88.3 (13.5)
43.3 (4.8)†
0
* − P < 0.05 between all groups; † − P < 0.05 between all groups except between scuba divers and attendants
THE DIVES
Closed circuit rebreather (CCR) dive: this was a wet dive
where the divers (n = 17) used a CCR (LAR VII, Draeger®)
and breathed 100% oxygen. Maximal pressure at depth was
130 kPa (3 msw, pO2 approaching 130 kPa) and the dive
time was 60 min.
Semi-closed circuit rebreather (S-CCR) dive: this was a
wet chamber dive where the divers (n = 12) used a S-CCR
(SIVA 55, Carleton®) and breathed 60% oxygen and 40%
nitrogen. Maximal pressure at depth was 300 kPa (20 msw,
pO2 approaching 180 kPa) and bottom time was 47 min.
Decompression was done according to the Canadian diving
tables (DCIEM Table 1: 21 msw/50 min). Total dive time
was 58 min.
Scuba dive: this was a partial wet, partial dry dive where the
divers (n = 17) used scuba (Mk 25/S550, Scubapro®) while
breathing compressed air. Maximal pressure at depth was
600 kPa (50 msw, pO2 approaching 126 kPa) with a bottom
time of 14 min. Diving was done in the wet compartment
of our pressure chamber; for both S-CCR and scuba dives,
the water temperature in the wet chamber was 13−15OC.
Decompression according to an adapted DCIEM Table 1 (51
msw/25 min) was done in the dry compartment where the
divers breathed chamber air. The total dive time was 71 min.
During this study, we also measured FENO of the attendants
(n = 12) who stayed inside the dry compartment during this
whole dive. Their maximal pressure at depth was 560 kPa
(46 msw, pO2 approaching 118 kPa).
MEASUREMENT OF FENO
Before and directly after every dive, the FENO was measured
using an electrochemical hand-held NO analyser (Niox
Mino®, Aerocrine AB, Sweden). Compared to on-line NO
analysers, the measured FENO values using the Niox Mino®
are statistically the same.7 All measurements were done
according to the ATS/ERS guidelines with an expiratory
flow rate of 50 ± 5 ml.s-1.8 The divers and attendants were
not allowed to drink coffee, eat or smoke within 1 h before
any measurement.
STATISTICAL ANALYSES
The results are presented as mean and standard deviation
(SD). Regarding the FENO data, the Shapiro-Wilk (S-W)
test (STATA Manual Reference G-M; 2003. p. 231) showed
a non-normal distribution for one of the groups (chamber
attendants). Therefore, we log transformed the FENO data
after which the S-W test showed a normal distribution
for all groups. Differences between the log transformed
pre- and post-dive FENO values were analysed using the
paired Student’s t-test. A P-value < 0.05 was considered
statistically significant. Analyses were performed using Stata
SE software (StataCorp, version 9.2).
RESULTS
All FENO measurements fell within the normal range (5–25
ppb).We found no differences between the three dive groups
regarding height, weight and smoking history (i.e., packyears). There was a significant difference in age between
the groups, except between scuba divers and attendants. The
CCR divers were the youngest divers and the attendants the
oldest (Table 1).
All dives produced a small decrease in FENO, with the
greatest decrease after a CCR dive (-2.8 ppb) and the smallest
Table 2
FENO (ppb) pre- and post-diving, mean (SD) shown;
CCR – closed circuit rebreather; S-CCR – semi-closed circuit rebreather; * P < 0.01
FENO
CCR divers
(n = 17)
Pre
Post
16.4 (8.0) 13.6(7.5)*
S-CCR divers
Scuba divers
Attendants
(n = 12)
(n = 17)
(n = 12)
Pre
Post
Pre
Post
Pre
Post
16.2 (7.2) 13.6(6.3)* 17.1 (5.6) 16.1 (5.2) 17.7 (9.8) 17.3 (9.1)
http://archive.rubicon-foundation.org
6
Diving and Hyperbaric Medicine Volume 40 No. 1 March 2010
in the attendants (-0.4 ppb). Only after a CCR or S-CCR dive
did this decrease in FENO become statistically significant at
the P < 0.05 level (Table 2).
Discussion
The results of the CCR and S-CCR groups, with a small
decrease in FENO, are in line with results from earlier
studies.2,6 As the CCR and S-CCR divers were the only
ones who performed a totally wet dive, submersion could
play a role in this reduction. During submersion there
is a central pooling of blood within the thoracic cavity.
This thoracic pooling results in an increased pulmonary
blood flow which leads to an increase in pulmonary artery
pressure.9 It is known that pulmonary arterial hypertension
results in decreased FENO, so it is conceivable that increased
pulmonary artery pressure could cause a drop in FENO.10
Secondly thoracic pooling leads to an improved ventilationperfusion relationship.9 This improvement could result in a
higher level of NO diffusion and, therefore, to a lower net
FENO. Eventually, both mechanisms could cause a more
pronounced decrease in FENO in a wet dive compared to a
dry hyperbaric chamber dive.
However, compared to earlier studies, in which only dry
chamber dives were performed, our findings showed a
smaller decrease in FENO.2,6 Therefore, submersion alone
cannot fully explain the decrease in FENO we found. More
plausible explanations for this decrease were given by
Puthucheary et al who stated that hyperbaric oxygen inhibits
iNOS which leads to a decrease in FENO.2 Also, the presence
of reactive oxygen species could scavenge NO, resulting in
decreased FENO.2
As FENO is a biological marker, normal deviations play a
role and must be taken into account. A difference of up to
2 ppb between two measurements of FENO can be found,
so a difference of more than 4 ppb is considered to be of
biological significance.11 Regarding the Niox Mino®, which
has an accuracy of +/- 2.5 ppb, a difference of more than 5
ppb is regarded as biologically significant.7 In view of this,
we conclude that, although we found a statistically significant
reduction of FENO in the CCR and S-CCR groups, these
minor changes are not bio-medically relevant.
also only measured changes of FENO in the conductive
compartment.2,4−6 To differentiate between the alveolar
and conductive compartments, the multiple exhalation
flow technique (MEFT) should be used.14 Since hyperbaric
hyperoxia produces alveolar damage, one should use
expiratory flow rates of at least 250 ml.sec-1, and we strongly
recommend that future studies use the MEFT to differentiate
between FENO changes in these two compartments after
exposure to an increased level of pO2.
Appendix
The FENO pathway is often visualized as a two-compartment
model with a conductive (airway to generation 17)
and alveolar compartment (generation 18 to alveolus).
FENO is a net result of flux and diffusion of NO in these
compartments.13 Based on this idea it is possible to calculate
the FENO using the formula of George et al.13
FENO = Caw,no + (Calv,no – Caw,no)*exp(–Daw,no/V)
where Caw,no is the airway wall concentration of NO, Calv,no
the steady-state alveolar concentration of NO, Daw,no diffusing
capacity of NO, and V is the exhalation flow rate.
•
•
•
FENO is not influenced by age, day-to-day or within-day
variations but is reduced in females.8,11
Acknowledgements
We would like to thank our participating military divers
and Willard van Ooij, MSc, for his additional statistical
advice.
References
1
Finally a limitation of the present study should be mentioned.
We measured the FENO with an expiratory flow rate of 50 ±
5 ml.sec-1, according to the ATS/ERS guidelines.8 At these
flow rates, one measures the FENO from the bronchus down
to the alveolus, but not the alveolus itself.12 To measure
the alveolar compartment FENO, the subject should exhale
in a controlled fashion over 8–10 sec at a flow rate of at
least 250 ml.sec-1, and sidestream sampling (of alveolar
gases) occurs during the final part of exhalation.10,13 As
we used a flow rate of 50 ml.sec-1, we measured changes in
the conductive compartment of the lungs only and not in
the alveolar compartment (see Appendix). Earlier studies
used flow rates of up to 100 ml.sec-1, implying that they
Healthy persons have a F ENO between 5 and 25
ppb.7,11,12
Values above 50 ppb can be found in exacerbation of
asthma and chronic obstructive pulmonary disease or
an acute eosinophilic airway inflammation.12
Values below 5 ppb can be found in smokers or after
strenuous efforts.8
2
3
4
5
Ten Hacken NH, Timens W, van der Mark TW, Nijkamp
FP, Postma DS, Folkerts G. [Nitric oxide and asthma]. Ned
Tijdschr Geneesk. 1999;143:1606-11. Dutch.
Puthucheary ZA, Liu J, Bennett M, Trytko B, Chow S, Thomas
PS. Exhaled nitric oxide is decreased by exposure to the
hyperbaric oxygen therapy environment. Mediators Inflamm.
2006;2006(5):72620, doi:10.1155/MI/2006/72620. PubMed
PMID: 17392577.
Clark JM, Lambertsen CJ. Pulmonary oxygen toxicity: a
review. Pharmacol Rev. 1971;23:38-133.
Lemaître F, Meunier N, Bedu M. Effect of air diving exposure
generally encountered by recreational divers: Oxidative stress?
Undersea Hyperb Med. 2002;29:39-49.
Schmetterer L, Strenn K, Kastner J, Eichler HG, Wolzt M.
Exhaled NO during graded changes in inhaled oxygen in man.
http://archive.rubicon-foundation.org
Diving and Hyperbaric Medicine Volume 40 No.1 March 2010
6
7
8
9
10
11
12
13
Thorax. 1997;52:736-8.
Taraldsøy T, Bolann BJ, Thorsen E. Reduced nitric oxide
concentration in exhaled gas after exposure to hyperbaric
hyperoxia. Undersea Hyperb Med. 2007;34:321-7.
Alving K, Janson C, Nordvall I. Performance of a new handheld device for exhaled nitric oxide measurements in adults
and children. Respir Res. 2006;7:67, doi: 10.1186/1465-99217-67. Pubmed PMID: 16626491.
ATS/ERS recommendations for standardized procedures
for the online and offline measurements of exhaled lower
respiratory nitric oxide and nasal nitric oxide. Am J Crit Care
Med. 2005;171:912-30.
Pendergast DR, Lundgren CEG. The underwater environment:
cardiopulmonary, thermal, and energetic demands. J Appl
Physiol. 2009;106: 76-83.
Geigel EJ, Hyde RW, Perillo IB, Torres A, Perkins PT,
Pietropaoli AP, et al. Rate of nitric oxide production by
lower alveolar airways of human lungs. J Appl Physiol.
1999;86:211-21.
Kharitonov SA, Gonio F, Kelly C, Meah S, Barnes PJ.
Reproducibility of exhaled nitric oxide measurements in
healthy and asthmatic adults and children. Eur Respir J.
2003;21:433-8.
Hemmingsson T, Linnarsson A, Gambert R. Novel hand-held
device for exhaled nitric oxide-analysis in research and clinical
applications. J Clin Monit. 2004;18:379-87.
George SC, Hogman M, Permutt S, Silkoff PE. Modeling
pulmonary nitric oxide exchange. J Appl Physiol. 2004;96:8319.
7
14 Kharitonov SA, Barnes PJ. Exhaled biomarkers. Chest.
2006;130:1541-6.
Submitted: 30 July 2009
Accepted: 03 December 2009
Pieter-Jan van Ooij, MD, is a senior diving medicine
physician at the Diving Medical Centre, Royal Netherlands
Navy.
Antoinette Houtkooper is a respiratory function technician
and head of the Ergometry and Respiratory Function Unit
at the Diving Medical Centre.
Rob van Hulst, MD, PhD, is Surgeon Captain (Navy) and
Head of the Diving Medical Centre.
Address for correspondence:
Pieter-Jan van Ooij
Diving Medical Centre, Royal Netherlands Navy
PO Box 10.000
1780 CA Den Helder
The Netherlands
Phone: +31-(0)223-653076
Fax: +31-(0)223-653148
E-mail: <[email protected]>